The preferred method today for conserving biological material while simultaneously maintaining its viability is deep freezing, followed by storage at temperatures below -30.degree. C. Cells such as spermatozoa, fertilized or unfertilized eggs, animal and plant cells cultivated in vitro, tissue samples, blood and other biological material can thus be stored over practically unlimited times and, if desired, brought back to life by thawing. The method is important in biological research, in animal and plant breeding and in the implanting of fertilized eggs or embryos, as practised recently in human medicine and animal husbandry.
When freezing living biological material, it is very important to lower the temperature regularly and at a rate at which any damage to the cells and their components is avoided. Damage can happen at too high (supraoptimal) or too low (suboptimal) rates of temperature reduction. Damage at supraoptimal cooling rates is very probably produced by development of ice crystals inside the cell and by the expansion of the crystals during the reheating phase (see P. Mazur: The role of intracellular freezing in the death of cells cooled at supraoptimal rates, Cryobiology 14, 251-272, 1977). Damage at suboptimal cooling rates can be explained by so-called solution effects (see P. Mazur: Slow freezing injury in mammalian cells, in "The Freezing of Mammalian Embryos", CIBA Foundation Symposium 52, 19-48, 1979). Safe methods for freezing biological samples therefore need a temperature program, exactly to be controlled, which depends on the type of material and the size of the sample.
The type of vessel in which the material is enclosed depends on the size of the sample as well. For greater quantities of material ampullae, preferably made of plastics, can be used, whose capacity is between about 2 and 10 milliliters, or tubes with a capacity of 0.25 to 0.5 milliliters; for single cells, sperm cells and embryos, thin plastic tubes, so-called "straws" with a diameter of 1.95 millimeters and a length of about 14 centimeters are preferred. Together with their surrounding liquid the specimen can be sucked into the straw, which, after this, is closed on both its ends. Because of their small volume, these straws allow an exact temperature control with minimal time lag.
Devices for deep freezing of biological samples of different description are commercially available. In one type of the known devices the sample vessels are kept in a bath made with a non-freezing liquid, such as freon, alcohol, methanol or isopentane, whereby the temperature of this bath is kept uniform by stirring or by circulation. In another type of apparatus the sample vessels are kept in a space with a circulating gas, which is held at a temperature as even as possible. The volume of liquid or gas is cooled by heat exchange with a cryogen, such as liquid nitrogen, or by a conventional freezing machine. A controlled heat source is used to keep the gaseous or liquid bath at the desired exact temperature. For this latter operation additional energy has to be spent, as well as for the amount of cryogen lost by evaporation.
By its nature this type of apparatus is relatively heavy and needs a lot of energy. A high expenditure of weight, volume and energy has to be made, only to cool and freeze a relatively small sample. Smaller apparatus with a better use of weight, volume and energy is therefore highly desirable.